Genetic Diversity Evaluation of Moringa oleifera, Lam from East Flores Regency
Using Marker Random Amplified Polymorphic DNA (RAPD) and Its Relationship to
Chemical Composition and In Vitro Gas Production
Markus Miten Kleden1,3*), Hendrawan Soetanto1), Kusmartono1) and Kuswanto2)
1) Faculty of Animal Husbandry, University of Brawijaya Jl. Veteran Malang 65145 East Java Indonesia
2) Faculty of Agriculture, University of Brawijaya, Malang, Indonesia
3) Faculty of Animal Husbandry, University of Nusa Cendana, Kupang, East Nusa Tenggara, Indonesia *) Corresponding author E-mail: mkleden21@gmail.com
Received: August 2, 2016 /Accepted: March 13, 2017
ABSTRACT
The research objective was to evaluate the genetic diversity of Moringa oleifera, Lam(MO) and its re-lationship to chemical composition and in vitro gas production (IVGP). Fresh MO leaves were kept fro-zen in ice gels pack until laboratory analysis. Four methods applied: RAPD marker for measuring DNA
concentration and purification; Kjeldhal and HPLC
for analysing proximate and amino acid (AA)
com-position; and IVGP. MO’s four distinct morpholo -gy found: green, red, reddish green and aromatic green. RAPD result analysis was 68.8-74.7 %, it means those MO had a close genetic similarity. The morphological differences are also related to leaves chemical composition variation. The high-est protein and AAs content were found in aro-matic green MO. Total IVGP at 96 hours reached
95.9, 99.3, 111, 115 mL per 500 mg DM in aromatic
green, green, reddish green, red MO, respectively
and statistically among those was highly significant difference (P<0.01). However, DM and OM digest
-ibility did not differ significantly and estimated ME
contents were similar suggesting MO leaves had
sufficient fermentable nitrogen amount required
to ensure rumen microbes normal activities. Con-clusively, those MO has a close genetic
relation-ship but the aromatic green MO more beneficial
due its higher content of crude protein and AAs.
Keywords: amino acid content; in vitro digestibility; miracle tree; morphological variation
INTRODUCTION
The ubiqitous spread of Moringa oleifera (MO) in Indonesia may have been as long as the migration history of Indonesian anchestor from
India and Indochina. This notion stems from the fact that the plant is originated from India but nowadays it has been endemic plant found elsewhere from the low to the highland of tropical regions (Bayé-Niwah
& Mapongmetsem, 2014). MO has many local names such as kelor (in Java island); maronggi
(Madura island), moltong (Flores island), hau fo (West Timor) and it is generally perceived by people in certain parts of Indonesia as anti black magic trees rather than as source of nutritious vegetables or fodder consumed by either human or animals. Nevertheless, in the last decade many studies on
MO’s nutritive values and its benefits for livestock
feed resources have been accumulated. Soetanto,
& Firsoni (2008) reported a significant increase
in in vitro organic matter digestibility and rumen microbial biomass production when MO was added to the diet consited of maize stover, commercial
concentrate and urea mollases-block. In other field
experiment Soetanto, Chuzaemi, & Marhaeniyanto
(2010) revealed that adding MO in the basal diet
of Ettawah goats had 83 % more weight gain that can be attributed to MO supplementation. Other authors reported a 14.5 % increase in daily weight gain of fat-tailed growing rams when MO and Samanea saman leaves were supplemented to a diet containing 18 % crude protein (Marhaeniyanto,
Soetanto, Kusmartono, & Hartutik, 2013). Recently, Dahlanuddin, Yanuarianto, Poppi, McLennan, & Quigley (2014) fed male Bali cattle aged between
6 and 12 months with four tree fodder hay as a sole
diet, i.e. Sesbania, Leucaena, MO and Gliricidia and revealed that only the first three fodder hay could increase the body weight from 0.47 to 0.22 kg
per head per day while gliricidia hay as the sole diet
seemed to satisfy the maintenance requirement only.
Cite this as: Kleden, M. M., Soetanto, H., Kusmartono, & Kuswanto. (2017). Genetic diversity evaluation of Moringa oleifera, Lam from East Flores Regency using marker Random Amplified Polymorphic DNA (RAPD) and its relationship to chemical composition and in vitro gas production. AGRIVITA Journal of Agricultural Science, 39(2), 219–231. http:// doi.org/10.17503/agrivita.v39i2.1027
The potential of MO as supplement for sheep has been reported by Mulyati, Kusmartono, Hartutik, &
Rusdi (2015) where the daily gain reached 107 g per
head per day. There was evidence demonstrated by
Rahardja, Fattah, & Toleng (2010) that reproductive performance improved in beefi cattle given MO in the
diet. For this reason it is not surprising if MO has been used widely for human and livestock nutrition due to
its uniqueness of nutrient composition that satisfy the most requirement of essential nutrients (Price, 2007), such as essential amino acids, minerals
and other antioxidant compounds (Nouman et al.,
2014). According to Ali (2014) altough MO contains
phenolic acids (gallic, chlorogenic, ellagic and
ferulic acid) and flavonoids (kaempferol, quercetin
and rutin) there is no evidence of harmful effect of feeding MO reported in the literature.
The main mineral contents of Na, Fe, Mn,
and Zn are about 192.95 ± 4.4; 107.48 ± 8.2; 81.65 ± 2.3; and 60.06 ± 0.3 ppm, respectively (Ogbe & Affiku, 2011). It therefore is likely that MO can be
used as the source of indispensable minerals for animal and human beings (Dasola, Tunbosun,
Adeyemi, & Abidemi, 2014). MO also called a
life-tree since it can be used as a source of food to overcome malnutrition (Sixl-Daniell, Sixl, W., Sixl,
G., & Fuchs, 2011; Azeez et al., 2013), as nutritional
supplement for weaning infant and nursing mother
(Kesharwani, Prasad, Roy, & Sahu, 2014) and
having anti fungal properties (Patel, P., Patel, N.,
Patel, D., Desai, & Meshram, 2014). Nowadays, the
plant has a highly value and cultivated largely in the tropics and sub-tropical areas. The plant cultivation is done in order to be used for food, herbs or medicine, and industrial objectives (Moyo, Masika,
Hugo, & Muchenje, 2011; Anwar, Latif, Ashraf, & Gilani, 2007). MO seed can be used as coagulant for water purification (Dalen, Pam, Izang, & Ekele, 2009; Padmapriya, Thamaraiselvi, Nivethini, & Thirunalasundari, 2014), and the leaf can be used
to increase blood haemoglobin (Hb) concentration, when it is used for feeding (Otitoju, Nwamarah,
Otitoju, Okorie, Stevens, & Baiyeri, 2014). The
richness of MO leaves in essential amino acids, vitamin A and minerals such as Na, Fe, Mn and Zn
(Ogbe & Affiku, 2011) may be used as a strategy to eliminate nutrient deficiencies for human as well as
for animals in certain regions such as East Nusa Tenggara where a long dry season causes poor growth of ruminant animals. Unfortunately, most people in East Nusa Tenggara do not aware of the
nutritive values of MO other than recognize it as a natural vegetable source for their daily consumption. In Flores island and other parts of East Nusa Tenggara, MO can be easily found anywhere and it usually grows surrounding house yard and dry land without irrigation. Nonetheless, the information on MO genetic diversity from this area is scarce and people generally descriminate them mainly based on the morphological differences of leaf petioles
color and fragrance exposure that may reflect their
genetic variability and adaptation to environmental conditions. Nevertheless, with the advancement of technology, genetic variation of plants can be determined more accurately using DNA-based
analysis such as random amplified polymorphic
DNA (RAPD) and,hence, it allows the application for plant breeding and selection as has been reported
by Rufai et al. (2013) in MO for human food and
animal fodder.
The objectives of this study were to ascertain the genetic diversity of MO in East Flores regency using marker RAPD and its relationship to the chemical composition and in vitro gas production.
MATERIALS AND METHODS MO Leaves Collection
Samples of fresh MO leaves were collected
from March to June 2014 on the basis of four
distinctive colour differences growing at the
village of Lewolere, district of Larantuka, East
Flores Regency, East Nusa Tenggara Province at
08’20’’50.9 SL and 122’57’53.5 EL, at 50 m above
sea level (Fig. 1).
The fresh leaves were placed in the plastic bags containing wet cotton layers to prevent water loss from the leaf cells, then put in the box cotaining iced gels and transported immediately to the central life science laboratory of University of Brawijaya for further DNA analysis. While other fresh leaves were sun dried under the shed and ground through a 1 mm sieve diameter for protein and amino acids analysis.
Isolation and DNA Purification
DNA isolation was carried out according to the
modified method of Doyle, J. J. & Doyle, J. L. (1990) on the fresh leaves samples as follow: weighing 0.2
g fresh kelor leaves and wrapped with aluminium
foil and stored at -20 0C. While frozen it was grinded
in the sterilized mortar and added liquid nitrogen and 500 µl CTAB buffer solution, decanted into 1.5
Site of MO sampling
District of Larantuka, East Flores Regency East Nusa Tenggara Province
Fig. 1. The site of MO collection ml sterilized plastic tube, gently mixed and covered
with parafin and incubated in the water bath at 65 0C
for 30 minutes. They were then centrifuged at 15493 g for 10 minute at 25 0C. The supernantan was
transfrred into 1.5 ml sterilized tubes, added with
aliquot volume of chloroform, and re-centrifuged
at 15493 g for 5 minute. The same procedure was repeated as in step 2 but chloroform was replaced by ammonium acetate and vortexed prior adding 2.5 times the volume with absolute ethanol, mixed until a white treath like form appereared. They
were then stored overnight in the -20 0C freezer,
centrifuges at 15493 g for 15 minutes at 4 0C until
pelleted DNA was formed. 500 µl 70 % ethanol was
added to the pelleted DNA and centrifuges at 15493
g for 10 minutes at 4 0C. The pelleted DNA was then
air dried , added with 50 µl TE buffer (pH=7.6) and stored at -20 0C for further analysis.
RAPD PCR Amplification
PCR amplification was performed using Go Taq Green PCR Mix kits (Promega) as follow:
mixing 1 µl Primer 10 pmol µl-1 with 3 µl double
destilled water, 5 µl PCR mixture and 1 µl DNA.
An initial denaturation temperature of 95 0C for 3 minutes and denaturation for 1 minute, annealing at 37 0C for 1 minute, extensions at 72 0C for 2 minutes and last extension at 72 0C for 5 minutes and back
to 95 0C for 45 cycles.
DNA fragment pattern produced were translated in binary data. The data, then, were processed and analyzed using Multi Variate Statistical Package (MVSP) with method of UPGMA (Unweight Pair Grouping Method with Arithmetic Averaging) (Sokal & Michener, 1958).
Chemical Analysis
Samples of MO leaves collected from the area under study were sun dried under the shed canopy and ground to pass the 2 mm size sieve prior
to proximate analysis (AOAC, 1990) particularly
crude protein content of MO leaves determined by Kjeldhal method.
Analysis of Amino Acid
Amino acid content of each sample of MO
leaves was determined by HPLC method (Lookhart
& Jones, 1985) on the basis of:
Remarks:
AA = Amino Acid Mr = relative masses
In Vitro Gas Production
Sample preparation and measurement of total gas production protocol were essentially according to the method of Makkar, Blümmel, & Becker (1995).
Rumen liquor was obtained from a Frisien Holstein cross bred cow which is equipped with a rumen
cannulae and fed with fresh elephant grass and
concentrate mixture in proportion of 60:40 to meet the above maintenance requirement. The animal was kept at the field laboratory of University of
Brawijaya and the using of this animal for experiment has obtained a permit from the ethical commission
of University of Brawijaya. A mixture of rumen fluid
and digesta was taken by aspirating it directly from the cannulae and placed it in a pre-warmed thermo
flask and carried to the nutrition laboratory where in
vitro gas production measurement was performed.
A mixture of rumen fluid and digesta was screened using four layer cheese cloth under continous flow
of CO2 gas. Approximately 340 ml screened rumen
fluid was added to mineral buffer to get 1500 ml of solution. 50 ml of rumen fluid buffer mixture was
added to each syringe containing approximately
500 mg of MO samples, blank and standard,
respectively and they were incubated at 39 0C in the waterbath to measure gas production at time
intervals 0,2,4,6,8,12,16,24,36,48,72, and 96 hours.
The data of each parameter was estimated
using exponential equation model according to
Ørskov & McDonald (1979), with a Neway computer packet program (Boga, Yurtseven, Kilic, Aydemir, &
Polat, 2014) according to exponential equation: Y = a + b (1-ect)
Where:
Y = amount of gas produced at t time
a = amount of gas produced by soluble fraction (ml per 500 mg DM)
b = amount of gas produced by insoluble fraction (ml per 500 mg of DM)
c = constant of gas production from insoluble
fraction (ml per hour)
a + b = total production of gas from fermented fraction (ml per 500 mg DM)
t = time of incubation (hour) e = 2.72 (natural logarithm)
Digestibilities of DM and OM were calculated as differences between samples and residues obatained after 96 hours incubation. All data obtained from IVGP was subjected to statistical analysis using astatistical package program of
SPSS version 18 (Levesque, 2007). RESULTS AND DISCUSSION
Morphological Differences and Genetic Diversity
There were four distinctive morphological differences of MO trees growing in the area of study based on the colours of petiole and leave odor (Fig. 2).This is also supported by evidence that in nature each type showed genetic diversity as demonstrated by the results of RAPD analysis. In general the purity of DNA analysis may be interfered by the presence of polysaccharides and polyphenolic compounds
(Lutz, Wang, Zdepski, & Michael, 2011). This
interference may be reduced by addition of CTAB as an ihibitor compound. This study showed that the purity of DNA found from the samples were high as indicated by the ratio of UV absorbance wave
length at Å260/Å280 which denotes as purity fell between 1.88 and 2.07, suggesting that neither
polysachharides nor polyphenolic interfered the purity of DNA (Table 1).
The result of PCR analysis using RAPD
marker demonstrated that from 20 OPA primers
only 18 resulted reproducible bands. A total of 79
DNA fragments had succesfully been amplified from
the genome of four MO samples resulting in 44
polimorphyc band or equal to 55.9 % polimorphic. Number of amplified fragments per primer varied between 250 bp and 2500 bp as shown in Fig. 3.
This indicates that among four MO samples there is
different sequence of DNA from the DNA fragment being amplified. Table 2 depicts the sequence of
each primer, the number of band and polimorphism percentage.
The percentage of polymorphic of four MO accessesion in this study was only 55.9 % which is considered lower than those reported by other authors such as Ojuederie, Igwe, Okuofu, & Faloye
(2013) who reported polymorphism of 10 MO from Western Nigeria was 81.5 % while Popoola (2014) found the corresponding figure for 13 MO accession in Nigeria was 77.9 %. Nevertheless, this finding
A. Green MO B. Reddish green MO
C. Red MO D. Aromatic green MO
Fig. 2. The differences of MO trees growing in the area of study based on the colours of petiole and leave odor is higher if compared by the report of Rufai et al.
(2013) that found only 32.7 % from five accessions
of MO in Malaysia. Furthermore, following a MVSP analysis of DNA fragment pattern using UPGMA (Sokal & Michener, 1958) the four MO accessions were found having close genetic similarity ranged between 68.8 % and 74.7 % (Fig. 4) suggesting that in the area under study the existing MO trees
might come from similar genus but due to genetic and environment interaction they differ phenotypic in petiole colors and odor shown in Fig. 2 table 2.
These differences may be reflected in chemical composition such as CP and AAs profile that are beneficial for selection of MO to produce a source
of essential nutrition either for human or animals.
Table 1. DNA concentration of MO from East Flores, Indonesia
No Sample Name Absorbance (A0) Concentration (ng µl-1)
260 280 Purity 1 A1 18.1 9.30 1.95 907 2 A2 9.10 4.69 1.94 454 3 B1 21.5 10.4 2.07 1,072 4 B2 8.66 4.37 1.98 433 5 C1 8.56 4.34 1.97 428 6 C2 7.42 3.72 1.99 371 7 D1 13 6.55 1.99 651 8 D2 10.8 5.73 1.88 538
Remarks: Samples of A1 and A2 = green MO; B1 and B2 = reddish green MO; C1 and C2 = red MO; D1 and D2 = aromatic green MO
2500
1500
1000
500
250
Fig. 3. Example of RAPD analysis of MO leaves using OPA10-OPA11 primer
Table 2. Sequence of 18 OPA primers with the number of scorable, amplified and polymorphic bands
Primer Name Sequence (5’-3’) Total Number of band Number of polymorphic band
Percentage of polymorphism
OPA 1 CAG GCC CTT C 5 2 40.0
OPA 2 TGC CGA GCT G 4 3 75.0
OPA 3 AGT CAG CCA C 4 0 0
OPA 4 AAT CGG GCT G 3 1 33.3
OPA 7 GAA ACG GGT G 2 2 100
OPA 8 GTG ACG TAG G 3 2 66.7
OPA 9 GGG TAA CGC C 4 2 50.0
OPA 10 GTG ATC GCA G 6 4 66.7
OPA 11 CAA TCG CCG T 6 6 100
OPA 12 TCG GCG ATA G 7 5 71.4
OPA 13 CAG CAC CCA C 5 1 20.0
OPA 14 TCT GTC CTG G 2 1 50.0
OPA 15 TTC CGA ACC C 3 2 66.7
OPA 16 AGC CAG CGA A 6 2 33.3
OPA 17 GAC CGC TTG T 4 3 75.0
OPA 18 AGG TGA CCG T 2 0 0
OPA 19 CAA ACG TCG G 8 7 87.5
OPA 20 GTT GCG ATC C 5 1 20.0
Total 79 44 55.9
68.8% 72.2% 74.7% A B C D
Simple Matching Coefficient
0.64 0.7 0.76 0.82 0.88 0.94 1
Fig. 4. Phylogenetic relationship among the studied MO accessions using UPGMA dendogram based on
RAPD Primary result OPA 1-20
Table 3. Proximate analysis of four accessions of MO leaves from East Flores
Items Morphology
Green Reddish Green Red Aromatic Green
Dry Matter (%) 88.8 87.2 88.3 86.2 Organic Matter (% DM) 89.1 90.1 91.3 88.3 Crude Protein (% DM) 33.9 27.5 28.4 36.5 Crude Fiber (% DM) 6.84 8.73 7.14 7.74 Crude Lipid (% DM) 7.42 9.99 8.99 6.97 NFE (% DM) 40.8 43.8 46.8 37.1 Ash (% DM) 10.9 9.92 8.70 11.7
Table 4. Amino acid profile of MO leaves varying in morphology (%)
Parameter Morphology
Green Reddish Green Red Aromatic Green
Amino Acid Essential
Arginine 1.59 1.09 1.11 1.94 Histidine 0.57 0.41 0.41 0.73 Leucine 2.20 1.68 1.70 2.70 Isoleucine 1.34 1.00 1.01 1.61 Lysine 1.38 1.04 1.05 1.47 Methionine 0.30 0.26 0.27 0.34 Threonine 1.11 0.82 0.83 1.43 Phenylalanine 1.77 1.32 1.34 2.17 Valine 1.61 1.19 1.21 1.91 Non Essential Aspartic Acid 3.03 2.00 2.03 3.89 Glutamic Acid 3.67 2.82 2.86 4.25 Serine 1.14 0.84 0.86 1.38 Glycine 1.23 0.89 0.91 1.39 Alanine 1.62 1.25 1.27 1.91 Tyrosine 1.64 1.15 1.17 1.75 Cystine 0.33 0.21 0.16 0.69 Proline 0.99 0.84 0.90 1.28
Table 5. In vitro gas production of MO leaves varying in morphology (ml per 500 mg DM)
Duration of Incubation
(hour)
Morphology
Green Reddish Green Red Aromatic Green SEM Prob.
2 4.22 5.07 4.92 4.55 0.191 0.619 4 10.9a 12.9b 13.0b 11.4a 0.545 0.021 8 27.3a 31.5b 31bc 28.3ac 1.02 0.034 12 47.7 52 51.3 47.9 1.12 0.080 16 61.3a 66b 66.8b 61.4a 1.47 0.026 24 76.4a 80.7b 84.6b 75.2a 2.16 0.011 36 85.7a 94.2b 98.5c 83.4a 3.56 0.000020 48 91.6a 102b 106c 87.9a 4.33 0.000003 72 96.8a 109b 112b 93.7a 4.46 0.000013 96 99.3a 111b 115b 95.9a 4.68 0.000045
Remarks: Values bearing different letter in the same row differ significantly at P<0.01. SEM = standard eror of mean; Prob. = probability
Chemical Composition and Amino Acids Profile Table 3 describes the result of proximate analysis of four MO accession leaves in which the aromatic green type was found containing the highest CP followed in descending order by green, red and reddish green types, respectively. In
contrast, the crude fiber (CF) content was relatively
similar among them. From the survey of literature, the CP content of MO leaves varies between 18.4
and 39.1 % (Ogbe & Affiku, 2011; Agbogidi & Ilondu, 2012; Madukwe, Ugwuoke, & Ezeugwu, 2013; Offor, Ehiri, & Njoku, 2014; Moyo, Masika, Hugo, & Muchenje, 2011; Mbailao, Mianpereum, & Albert, 2014; Melesse, 2011; Sodamade, Bolaji, & Adeboye, 2013). While for CF content the corresponding figures are 5.43 to 11.2 (Mbailao, Mianpereum, & Albert, 2014; Melesse, 2011; Ogbe & Affiku, 2011;
El-Sohaimy, Hamad, Mohamed, Amar, & Al-Hindi,
2015; Sodamade, Bolaji, & Adeboye, 2013).
The result of AA analysis presented in Table 4 does not include essential AA tryptophan due to limitation of our laboratory to obtain chemical standard for tryptophan and some non-essential AAs. Therefore, only 9 and 8 essential and non-essential AAs, respectively, are presented in Table 4.
In general, AA profile of aromatic green type
showed superior compared to the other three MO leaf type, even when compared with the results of
Moyo, Masika, Hugo, & Muchenje (2011) and Khalel et al. (2014). While for other types of MO leaves
contain either essential or non-essential AAs within the value range reported by many investigators
(Zaku, Emmanuel, Tukur, & Kabir, 2015; Mbailao, Mianpereum, & Albert, 2014; Nouman et al., 2014).
In Vitro Gas Production
There is generally agreed that protein content in feed affects rumen microbial activity in favor of
gas production. Melesse (2011) reported a positive
correlation between CP content and IVGP of two species of MO leaves. In contrast this current study was not in accordance with the report of Melesse
(2011) as the aromatic green MO type that contained
higher CP produced gas at a lesser volume than other MO leaf accessions (Table 3 and Table 5). In addition, there was a lag phase (Table 6 and Fig. 5) found in all MO types suggesting that there is an anti-nutritional factor e.g. tannin in the leaf affecting the commencement of microbial fermentation in the rumen. Total IVGP at 96 hours reached 95.9,
99.3, 111 and 115 ml per 500 mg DM in aromatic
green, green, reddish green and red MO leaves, respectively and statistically among those was highly
significant difference (P<0.01). Nevertheless, IVGP of this present finding fell within the gas production
ranges reported by previous studies (Asaolu,
Odeyinka, Binuomote, Odedire, & Babayemi, 2014; Karim, Amin, Moniruzzaman, Sarker, & Kabir, 2015; Melesse, 2011).
As shown in Table 6 both DM and OM digestibility
were surprisingly not significant difference among
them but higher than reproted by Nezarati, Nazer,
Ramin, & Abolfazi (2014) and hence the estimated
ME content was similar and addition of protein in the diet usually has catalytic effect on rumen microbial
activity if there is nutritional deficiency for microbial
growth leading to improvement in feed digestion
(Leng, 1993). However, this condition may not
always operate especially when the ratio of protein and carbohydrate is low.
Item
MO type
SEM Prob.
Green Reddish Green Red Aromatic Green
a (ml) -3.01 -2.3 -2.76 -2.73 0.147 0.689 b (ml) 106a 119b 125c 101a 5.62 0.000207 c (ml per hour) 0.044 0.041 0.039 0.047 0.002 0.065 a + b (ml) 109a 121b 128c 104a 5.56 0.000033 DMD (%) 63.3 66.8 67.1 64.0 1.13 0.595 OMD (%) 89.4 91.3 91.4 89.7 0.729 0.740 ME (MJ/kg DM)* 8.31 8.15 8.45 8.40 0.014 0.244
Fig. 5. In vitro gas production of different MO leaves collected from East Flores (ml per 500 mg DM)
Remarks: Values bearing different superscript in the same row differ significantly at P<0.01; * = ME estimated from equation ME = 2.2 + (0.136*Gv) + (0.057 x CP) (Makkar, 2002), where: ME = metabolizable energy; CP = crude protein in percent; and Gv = the net gas production in ml from 200 mg dry sample after 24 h of incubation, SEM = standard eror of mean; Prob. = probability
Table 6. Coefficient digestion of dry matter and organic matter of MO leaves
CONCLUSION
From this present study it can be concluded that MO found in East Flores Regency has a close genetic relation each other despite distinct morphological, crude protein and amino acids differences. Nevertheless, the difference in CP content did not affect in vitro DMD and OMD suggesting that MO leaves as a single ingredient
has sufficient amount of fermentable nitrogen
ensuring a normal activity of rumen microbes to digest food. Further studies under in vivo conditions on the effect of MO leaves supplementation are warranted.
ACKNOWLEDGEMENTS
This article is a part of the researches for doctoral degree in animal science at the Faculty of Animal Husbandry, University of Brawijaya,
Indonesia of the first author. The authors are thankful for the financial support provided by the Directorate
General of Higher Education, Ministry of Research Technology and Higher Education, Republic of Indonesia. Our sincere gratitude goes to Mr. Theodorus Niron for helping MO leaves collection and Mr. Sugiono for technical assistance given
during IVGP measurement. Lastly, we are indebted
for the tangible and professional assistance in RAPD
analysis given by Ms. Fitri of Central Laboratory for Life Sciences, University of Brawijaya.
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